32 Journal of PharmaSciTech Nuclear Magnetic Resonance (NMR) Spectroscopy in Protein Research 1 1 2 Shovanlal Gayen* , Nirmal Jayabalan , Souvik Basak Abstract Nuclear magnetic resonance spectroscopy is increasingly used in protein research. It can be used for protein structure determination in solution, close to the physiological environment. It can determine also the protein dynamics which is very important to understand biological phenomena. This article summarizes about the techniques and methods in NMR routinely used in protein research. Keywords: Nuclear Overhauser effect, NMR, Protein ISSN: 2231 3788 (Print) 2321 4376 (Online) Review Article 1 Department of Pharmaceutical Sciences, Dr Harisingh Gour University, Sagar 470003, MP, India 2 Dr B.C. Roy College of Pharmacy & Allied Health Sciences, Bidhan Nagar, Durgapur 713206, WB, India *Correspondence: slgayen@dhsgsu.in; Tel.: +91-8827680414 1.Introduction Biomolecular NMR spectroscopy has been proved to be an effective tool in modern biological research in determining macromolecular structures in solution and the dynamic properties of their structures 1 relevant to the biological functions . Recent developments (eg. uniform or specific isotopic labeling, transverse relaxation optimization spectroscopy [2] residual dipolar coupling etc.) make NMR spectroscopy an exciting technique in characterizing the larger systems including protein-protein complexes and protein-nucleic acid complexes. It has the power to detect very weak interaction at atomic resolution required for the modern drug discovery processes [3]. This study aims at highlighting the use of NMR spectroscopy in protein research. 2. Parameters in NMR 2.1. Chemical shift There are a number of observables in NMR that give information about molecular structure and dynamics. The most important one is the chemical shift. The static magnetic field B is not equal to the applied 0 magnetic field as electrons around the nucleus shield it from the applied field. Therefore the individual resonance frequencies are slightly different which reflects the different chemical environments. The resonance frequencies are termed as chemical shift. The unit of chemical shift is parts per million (ppm) in order to have chemical shift values independent of the static magnetic field. 2.2. J coupling J coupling is the coupling between the two nuclear spins due to the influence of bonding electrons on the magnetic field between the two nuclei. It is mediated through chemical bonds connecting the two spins. Depending on the spin state of scalar coupled spin the energy levels of each spin is slightly altered. This gives rise to the splitting of the resonance lines. J couplings give information regarding the dihedral angles that can be estimated through the Karplus curve. Scalar couplings are also used in the multidimensional experiments to transfer the magnetization of one spin to another in order to identify the spin systems. 2.3. Nuclear Overhauser effect NOESY (Nuclear Overhauser Effect Spectroscopy) spectra give inter- proton distance information in molecules if the protons are 5 Å or less apart in space. In order to calculate the structure of a protein many such distance information must be identified in an unambiguous fashion. The distance information is through space coupling and not through bond, because the dipolar cross relaxation depends on the distance between the interacting spins [1]. During analysis of a NOESY spectrum the integrated intensity of a given cross peak is interpreted in terms of the distance between the two protons giving rise to the peak. Accurate analysis requires the minimization of any effects that may systematically alter the intensity of peak. Choice of mixing time is particularly very important. Large molecules for example proteins generally give better NOEs at higher field. A midsize molecule (1000- 1500MW range) may have NOEs that are close to zero and a ROESY (rotating frame Overhauser effect spectroscopy) may be required to see those molecules [1]. 2.4. Residual dipolar couplings Residual dipolar couplings (RDC) not only give additional structural information but are very much important for defining long range interactions for example the relative orientation of two protein domains. The rapid tumbling of diamagnetic molecules in solutions makes it impossible to measure the potential directional information contained in the dipolar couplings. In order to obtain the orientation restraints from residual dipolar coupling measurements the protein molecule is weakly aligned in slightly anisotropic solutions for example; 5% mixture of DHPC/DMPC forming micelles in aqueous solution, filamentous phages. 2.5. NMR spectroscopy for larger molecules: TROSY NMR studies of larger size protein (more than 30 kDa) are mainly limited by the fact that they have larger correlation time or shorter transverse relaxation times. Signal decays more rapidly resulting in line broadening and poor resolution. A new NMR method called transverse relaxation optimized spectroscopy (TROSY) can overcome this line width problem [2]. This method extends the range of molecular size (up to 250 kDa) that can be studied by NMR. It has also been reported that TROSY-based triple resonance experiments are more sensitive than the conventional triple resonance experiments. However it generally requires high magnetic field strength. 3. Protein Structure determination by NMR Structure determination of biological macromolecules such as proteins and nucleic acids is very much important for understanding their biological function. Moreover, protein function depends on structural rearrangement or requires appropriate changes in the structure. Identification of the relationship between structure, flexibility and function is important in providing insights into structural biology. X-ray crystallography and Nuclear Magnetic Resonance (NMR) spectroscopy are the only two experimental methods for determination of high resolution structure of http://www.pharmascitech.in Volume 4, Issue 2, 2015; Journal of PharmaSciTech